Connexins 30 and 43 expression changes in relation to age-related hearing loss.

Age-related hearing loss (ARHL), also known as presbycusis, is the number one communication disorder for aging adults. Connexin proteins are essential for intercellular communication throughout the human body, including the cochlea. Mutations in connexin genes have been linked to human syndromic and nonsyndromic deafness; thus, we hypothesize that changes in connexin gene and protein expression with age are involved in the etiology of ARHL. Here, connexin gene and protein expression changes for CBA/CaJ mice at different ages were examined, and correlations were analyzed between the changes in expression levels and functional hearing measures, such as ABRs and DPOAEs. Moreover, we investigated potential treatment options for ARHL. Results showed significant downregulation of Cx30 and Cx43 gene expression and significant correlations between the degree of hearing loss and the changes in gene expression for both genes. Moreover, dose-dependent treatments utilizing cochlear cell lines showed that aldosterone hormone therapy significantly increased Cx expression. In vivo mouse treatments with aldosterone also showed protective effects on connexin expression in aging mice. Based on these functionally relevant findings, next steps can include more investigations of the mechanisms related to connexin family gap junction protein expression changes during ARHL; and expand knowledge of clinically-relevant treatment options by knowing what specific members of the Cx family and related inter-cellular proteins should be targeted therapeutically.

Upregulation of astroglial connexin 30 impairs hippocampal synaptic activity and recognition memory.

Astrocytes crucially contribute to synaptic physiology and information processing. One of their key characteristics is to express high levels of connexins (Cxs), the gap junction-forming protein. Among them, Cx30 displays specific properties since it is postnatally expressed and dynamically upregulated by neuronal activity and modulates cognitive processes by shaping synaptic and network activities, as recently shown in knockout mice. However, it remains unknown whether local and selective upregulation of Cx30 in postnatal astrocytes within a physiological range modulates neuronal activities in the hippocampus. We here show in mice that, whereas Cx30 upregulation increases the connectivity of astroglial networks, it decreases spontaneous and evoked synaptic transmission. This effect results from a reduced neuronal excitability and translates into an alteration in the induction of synaptic plasticity and an in vivo impairment in learning processes. Altogether, these results suggest that astroglial networks have a physiologically optimized size to appropriately regulate neuronal functions.

Recent insights into gap junction biogenesis in the cochlea.

In the cochlea, connexin 26 (Cx26) and connexin 30 (Cx30) co-assemble into two types of homomeric and heteromeric gap junctions between adjacent non-sensory epithelial cells. These channels provide a mechanical coupling between connected cells, and their activity is critical to maintain cochlear homeostasis. Many of the mutations in GJB2 or GJB6, which encode Cx26 and Cx30 in humans, impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. Thus, deciphering the connexin trafficking pathways in situ should represent a major step forward in understanding the pathogenic significance of many of these mutations. A growing body of evidence now suggests that Cx26/Cx30 heteromeric and Cx30 homomeric channels display distinct assembly mechanisms. Here, we review the most recent advances that have been made toward unraveling the biogenesis and stability of these gap junctions in the cochlea.

A Gap-Junction Mutation Reveals That Outer Hair Cell Extracellular Receptor Potentials Drive High-Frequency Cochlear Amplification.

Cochlear amplification enables the enormous dynamic range of hearing through amplifying cochlear responses to low- to moderate-level sounds and compressing them to loud sounds. Amplification is attributed to voltage-dependent electromotility of mechanosensory outer hair cells (OHCs) driven by changing voltages developed across their cell membranes. At low frequencies, these voltage changes are dominated by intracellular receptor potentials (RPs). However, OHC membranes have electrical low-pass filter properties that attenuate high-frequency RPs, which should potentially attenuate amplification of high-frequency cochlear responses and impede high-frequency hearing. We made in vivo intracellular and extracellular electrophysiological measurements from the organ of Corti of male and female mice of the CBA/J strain, with excellent high-frequency hearing, and from the CD-1 mouse strain, which has sensitive hearing below 12 kHz but loses high-frequency hearing within a few weeks postpartum. The CD-1 mouse strain was transfected with an A88V mutation of the connexin 30 gap-junction protein. By blocking the action of the GJ protein to reduce input resistance, the mutation increased the OHC extracellular RP (ERP) magnitude and rescued high-frequency hearing. However, by increasing the organ of Corti resistance, the mutation rescued high-frequency hearing through preserving the OHC extracellular RP (ERP) magnitude. We measured the voltage developed across the basolateral membranes of OHCs, which controls their electromotility, for low- to high-frequency sounds in male and female mice of the CD-1 strain that expressed the A88V mutation. We demonstrate that ERPs, not RPs, drive OHC motility and cochlear amplification at high frequencies because at high frequencies, ERPs are not frequency attenuated, exceed RPs in magnitude, and are appropriately timed to provide cochlear amplification.SIGNIFICANCE STATEMENT Cochlear amplification, which enables the enormous dynamic range of hearing, is attributed to voltage-dependent electromotility of the mechanosensory outer hair cells (OHCs) driven by sound-induced voltage changes across their membranes. OHC intracellular receptor potentials are electrically low-pass filtered, which should hinder high-frequency hearing. We measured the intracellular and extracellular voltages that control OHC electromotility in vivo in a mouse strain with impaired high-frequency hearing. A gap-junction mutation of the strain rescued high-frequency hearing, increased organ of Corti resistance, and preserved large OHC extracellular receptor potentials but reduced OHC intracellular receptor potentials and impaired low-frequency hearing. We concluded intracellular potentials drive OHC motility at low frequencies and extracellular receptor potentials drive OHC motility and cochlear amplification at high frequencies.

Microglia maintain the normal structure and function of the hippocampal astrocyte network.

Microglial control of activity-dependent plasticity and synaptic remodeling in neuronal networks has been the subject of intense research in the past several years. Although microglia-neuron interactions have been extensively studied, less is known about how microglia influence astrocyte-dependent control over neuronal structure and function. Here, we explored a role for microglia in regulating the structure and function of the astrocyte syncytium in mouse hippocampus. After depleting microglia using a CSF1R antagonist (PLX5622, Plexxikon), we observed severe disruption of astrocyte syncytial isopotentiality and dye coupling. A decrease in astrocyte-specific gap junction connexin (Cx) 30 and 43 expression, at least partially accounts for these microglia-dependent changes in astrocytes. Because neuronal function requires intact astrocyte coupling, we also evaluated the effects of microglia depletion on synaptic transmission in the hippocampus. Without microglia, the strength of synaptic transmission was reduced at baseline and after long-term potentiation (LTP). Conversely, priming microglia with systemic injections of lipopolysaccharide enhanced CA3-CA1 synaptic transmission. This microglia-induced scaling of synaptic transmission was associated with increased expression of post-synaptic scaffold proteins (Homer1) in CA1. However, astrocyte network function was not affected by microglia priming, indicating that microglia-dependent effects on astrocytes and neurons vary across functional states. Through manipulation of microglia in the brain, our results reveal the importance of microglia in homeostatic regulation of the astrocyte syncytium and scaling of synaptic transmission. These novel mechanisms uncover a new direction for future studies interrogating microglia function in various physiological and pathological contexts.

Decoupling astrocytes in adult mice impairs synaptic plasticity and spatial learning.

The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks by gap junction coupling, mainly composed of two gap junction channel proteins, connexin 30 (Cx30) and connexin 43 (Cx43). To circumvent developmental perturbations and to test whether astrocytic gap junction coupling is required for hippocampal neural circuit function and behavior, we generate and study inducible, astrocyte-specific Cx30 and Cx43 double knockouts. Surprisingly, disrupting astrocytic coupling in adult mice results in broad activation of astrocytes and microglia, without obvious signs of pathology. We show that hippocampal CA1 neuron excitability, excitatory synaptic transmission, and long-term potentiation are significantly affected. Moreover, behavioral inspection reveals deficits in sensorimotor performance and a complete lack of spatial learning and memory. Together, our findings establish that astrocytic connexins and an intact astroglial network in the adult brain are vital for neural homeostasis, plasticity, and spatial cognition.

Dynamic Spatiotemporal Expression Changes in Connexins of the Developing Primate's Cochlea.

Connexins are gap junction components that are essential for acquiring normal hearing ability. Up to 50% of congenital, autosomal-recessive, non-syndromic deafness can be attributed to variants in GJB2, the gene that encodes connexin 26. Gene therapies modifying the expression of connexins are a feasible treatment option for some patients with genetic hearing losses. However, the expression patterns of these proteins in the human fetus are not fully understood due to ethical concerns. Recently, the common marmoset was used as a primate animal model for the human fetus. In this study, we examined the expression patterns of connexin 26 and connexin 30 in the developing cochlea of this primate. Primate-specific spatiotemporal expression changes were revealed, which suggest the existence of primate-specific control of connexin expression patterns and specific functions of these gap junction proteins. Moreover, our results indicate that treatments for connexin-related hearing loss established in rodent models may not be appropriate for human patients, underscoring the importance of testing these treatments in primate models before applying them in human clinical trials.

Astrocyte-derived neurons provide excitatory input to the adult striatal circuitry.

Astrocytes have emerged as a potential source for new neurons in the adult mammalian brain. In mice, adult striatal neurogenesis can be stimulated by local damage, which recruits striatal astrocytes into a neurogenic program by suppression of active Notch signaling (J. P. Magnusson et al., Science 346, 237-241 [2014]). Here, we induced adult striatal neurogenesis in the intact mouse brain by the inhibition of Notch signaling in astrocytes. We show that most striatal astrocyte-derived neurons are confined to the anterior medial striatum, do not express established striatal neuronal markers, and exhibit dendritic spines, which are atypical for striatal interneurons. In contrast to striatal neurons generated during development, which are GABAergic or cholinergic, most adult astrocyte-derived striatal neurons possess distinct electrophysiological properties, constituting the only glutamatergic striatal population. Astrocyte-derived neurons integrate into the adult striatal microcircuitry, both receiving and providing synaptic input. The glutamatergic nature of these neurons has the potential to provide excitatory input to the striatal circuitry and may represent an efficient strategy to compensate for reduced neuronal activity caused by aging or lesion-induced neuronal loss.

Astrocytes close the mouse critical period for visual plasticity.

Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix-degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA-guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.

Astroglial Cx30 differentially impacts synaptic activity from hippocampal principal cells and interneurons.

Astrocytes play important roles in brain function via dynamic structural and functional interactions with neurons. Yet the underlying mechanisms remain poorly defined. A typical feature of astrocytes is the high expression of connexins, which mediate their extensive intercellular communication and regulate their structural properties. In particular, connexin 30 (Cx30), one of the two connexins abundantly expressed by astrocytes, was recently shown to be a critical regulator of excitatory synaptic transmission by controlling the astroglial coverage of synapses. However, the role of Cx30 in the regulation of inhibitory synaptic transmission and excitatory/inhibitory balance remains elusive. Here, we investigated the role of astroglial Cx30 on the electrophysiological and morphological properties of five classes of hippocampal CA1 stratum oriens and pyramidale neurons, defined by the unsupervised Ward's clustering. Using Cx30 knockout mice, we found that Cx30 alters specific properties of some subsets of CA1 interneurons, such as resting membrane potential and sag ratio, while other parameters, such as action potential threshold and saturation frequency, were more frequently altered among the different classes of neurons. The excitation-inhibition balance was also differentially and selectively modulated among the different neuron subtypes. Only slight morphological differences were observed on reconstructed neurons. Altogether, these data indicate that Cx30 differentially alters the electrophysiological and morphological properties of hippocampal cell populations, and modulates both their excitatory and inhibitory inputs. Astrocytes, via Cx30, are thus active modulators of both excitatory and inhibitory synapses in the hippocampus.

Polydisperse molecular architecture of connexin 26/30 heteromeric hemichannels revealed by atomic force microscopy imaging.

Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. Although homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy. For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of three Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements that can be derived from the law of total probabilities. Atomic force microscopy imaging of ringlike structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.

Cisplatin-induced ototoxicity in organotypic cochlear cultures occurs independent of gap junctional intercellular communication.

Cisplatin is a very effective chemotherapeutic, but severe and permanent hearing loss remains a prevalent side effect. The processes underpinning cisplatin-induced ototoxicity are not well understood. Gap junction channels composed of connexin (Cx) subunits allow for the passage of small molecules and ions between contacting neighboring cells. These specialized channels have been postulated to enhance cisplatin-induced cell death by spreading "death signals" throughout the supporting cells of the organ of Corti. This study sought to investigate the role of Cx43 in cisplatin-induced ototoxicity using organotypic cochlear cultures from control and two Cx43-mutant mouse strains harboring either a moderate (Cx43I130T/+) or severe (Cx43G60S/+) reduction of Cx43 function. Cochlear cultures from Cx43-mutant mice with a severe reduction in Cx43-based gap junctional intercellular communication (GJIC) had an enhanced number of hair cells that were positive for cleaved caspase 3, a marker of active apoptosis, after cisplatin treatment. In cisplatin-treated organotypic cochlear cultures, there was a decrease in the co-localization of Cx26 and Cx30 compared with untreated cultures, suggesting that cisplatin causes reorganization of connexin composition in supporting cells. Both Cx26 and Cx30 protein expression as well as GJIC were decreased in organotypic cochlear cultures treated with the gap-junction blocker carbenoxolone. When cisplatin and carbenoxolone were co-administered, there were no differences in hair cell loss compared with cisplatin treatment alone. Using cisplatin-treated control and Cx43-ablated organ of Corti derived HEI-OC1 mouse cells, we found that greatly reducing GJIC led to preferential induction of an ER stress pathway. Taken together, this study strongly suggests that inhibition of GJIC in organ of Corti cells does not lead to differential susceptibility to cisplatin-induced ototoxicity. Although cisplatin causes the same degree of cell death in gap junction competent and incompetent cochlear cells, the engagement of the mitochondrial dysregulation and ER stress differs.

Connexin 30 mediated rewiring of glucose metabolism in rat C6 xenograft and grades of glioma.

Connexin 30 (Cx30), a tumour-suppressive gap junctional protein, impacts on insulin-like growth factor receptor 1-mediated progression and stemness of glioma. Of late, metabolic reprogramming, a recently adjudged hall mark of malignancy, could reasonably associated with the changes in gap junctional communication in glioma. This newly recognized hallmark of reprogramming of metabolism to maintain the rapid proliferation necessitates further probing to establish the stronger hall marks. Hence, the current study attempted to link the association between the expression of Cx30 with glucose uptake and glucose metabolism in glioma. We have transfected Cx30 in C6 glioma cells, characterized by a low level of intercellular communication and developed xenografts to study the status of glucose transporters (GLUTs), hexokinase 2 and Pyruvate dehydrogenase kinase 1 (PDK 1) along with human glioma tissues by RT-PCR and immunoblotting. The results showed a significant increase in the levels of GLUTs, hexokinase 2 and PDK 1 in C6-implanted rat xenografts and high grades compared to their respective controls, whereas Cx30-transfected C6-implanted rat xenograft and low grades show no significant change compared to that of controls supporting the association between Gap junctional communications and glucose metabolism. We strongly speculate the impact of Cx30 over the glucose metabolism that might provide therapeutic prospects and challenges for anti-glycolytic cancer therapy.

Vitamin D receptor and metabolite effects on corneal epithelial cell gap junction proteins.

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)2D3 and 24R,25(OH)2D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR-/-) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)2D3 or 24R,25(OH)2D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca++ (Ca++i) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)2D3 and 24R,25(OH)2D3. Cx30 and Cx43 protein levels were also significantly increased in VDR-/- MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)2D3 and 1,25(OH)2D3. Ca++i was not affected by 1,25(OH)2D3 or 24R,25(OH)2D3 in HCEC or MPCEC. We conclude that both 1,25(OH)2D3 and 24R,25(OH)2D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca++i.

Evidence of decreased gap junction coupling between astrocytes and oligodendrocytes in the anterior cingulate cortex of depressed suicides.

Glial dysfunction is a major pathophysiological feature of mood disorders. While altered astrocyte (AS) and oligodendrocyte-lineage (OL) functions have been associated with depression, the crosstalk between these glial cell types has never been assessed in that context. AS are potent regulators of myelination, in part through gap junction (GJ) channels formed by the heterotypic coupling of AS-specific (Cx30 and Cx43) and OL-specific (Cx32 and Cx47) connexins. This study therefore aimed at addressing the integrity of AS/OL coupling in the anterior cingulate cortex (ACC) of depressed suicides. Using immunofluorescence and confocal imaging, we characterized the distribution of Cx30 and mapped its expression onto OL somas, myelinated axons, and brain vasculature in postmortem brain samples from depressed suicides (N = 48) and matched controls (N = 23). Differential gene expression of key components of the GJ nexus was also screened through RNA-sequencing previously generated by our group, and validated by quantitative real-time PCR. We show that Cx30 expression localized onto OL cells and myelinated fibers is decreased in deep cortical layers of the ACC in male-depressed suicides. This effect was associated with decreased expression of OL-specific connexins, as well as the downregulation of major connexin-interacting proteins essential for the scaffolding, trafficking, and function of GJs. These results provide a first evidence of impaired AS/OL GJ-mediated communication in the ACC of individuals with mood disorders. These changes in glial coupling are likely to have significant impact on brain function, and may contribute to the altered OL function previously reported in this brain region.

Connexin30 and Connexin43 show a time-of-day dependent expression in the mouse suprachiasmatic nucleus and modulate rhythmic locomotor activity in the context of chronodisruption.

BACKGROUND: The astroglial connexins Cx30 and Cx43 contribute to many important CNS functions including cognitive behaviour, motoric capacity and regulation of the sleep-wake cycle. The sleep wake cycle, is controlled by the circadian system. The central circadian rhythm generator resides in the suprachiasmatic nucleus (SCN). SCN neurons are tightly coupled in order to generate a coherent circadian rhythm. The SCN receives excitatory glutamatergic input from the retina which mediates entrainment of the circadian system to the environmental light-dark cycle. Connexins play an important role in electric coupling of SCN neurons and astrocytic-neuronal signalling that regulates rhythmic SCN neuronal activity. However, little is known about the regulation of Cx30 and Cx43 expression in the SCN, and the role of these connexins in light entrainment of the circadian system and in circadian rhythm generation. METHODS: We analysed time-of-day dependent as well as circadian expression of Cx30 and Cx43 mRNA and protein in the mouse SCN by means of qPCR and immunohistochemistry. Moreover, we analysed rhythmic spontaneous locomotor activity in mice with a targeted deletion of Cx30 and astrocyte specific deletion of Cx43 (DKO) in different light regimes by means of on-cage infrared detectors. RESULTS: Fluctuation of Cx30 protein expression is strongly dependent on the light-dark cycle whereas fluctuation of Cx43 protein expression persisted in constant darkness. DKO mice entrained to the light-dark cycle. However, re-entrainment after a phase delay was slightly impaired in DKO mice. Surprisingly, DKO mice were more resilient to chronodisruption. CONCLUSION: Circadian fluctuation of Cx30 and Cx43 protein expression in the SCN is differently regulated. Cx30 and astroglial Cx43 play a role in rhythm stability and re-entrainment under challenging conditions.

Dysfunction of astrocytic connexins 30 and 43 in the medial prefrontal cortex and hippocampus mediates depressive-like behaviours.

Astrocytic connexin dysfunction is closely associated with synaptic impairment and contributes to the pathological development of depressive-like behaviours. However, little is known about the expression of connexins in astrocytes from different brain regions, or how tissue specific connexin expression affects local neuronal activity. Here, we established a mouse model of chronic social defeated stress (CSDS), from which we isolated astrocytes from the medial prefrontal cortex (mPFC), hippocampus, amygdala, and ventral tegmental area (VTA). Expression profiling was then performed for connexins Cx26, Cx30, and Cx43. Expression of Cx30 and Cx43 was significantly decreased in mPFC and hippocampus of CSDS mice and was strongly associated with decreases in neuronal activity. Furthermore, overexpression of Cx30 and Cx43 in the mPFC and hippocampus increased neuronal activity and inhibited depressive-like behaviours; while suppression of Cx30 and Cx43 in normal mice was sufficient to reduce neuronal activity and induced depressive-like behaviours. Taken togetner, aberrant expression of astrocytic Cx30 and Cx43 in the mPFC and hippocampus significantly affects brian region-specific neuronal activity and drives depressive-like behaviours. These observations provide novel insights into the role of astrocyte gene expression in stress-induced depressive-like behaviours.

Early Hearing Loss upon Disruption of Slc4a10 in C57BL/6 Mice.

The unique composition of the endolymph with a high extracellular K+ concentration is essential for sensory transduction in the inner ear. It is secreted by a specialized epithelium, the stria vascularis, that is connected to the fibrocyte meshwork of the spiral ligament in the lateral wall of the cochlea via gap junctions. In this study, we show that in mice the expression of the bicarbonate transporter Slc4a10/Ncbe/Nbcn2 in spiral ligament fibrocytes starts shortly before hearing onset. Its disruption in a C57BL/6 background results in early onset progressive hearing loss. This hearing loss is characterized by a reduced endocochlear potential from hearing onset onward and progressive degeneration of outer hair cells. Notably, the expression of a related bicarbonate transporter, i.e., Slc4a7/Nbcn1, is also lost in spiral ligament fibrocytes of Slc4a10 knockout mice. The histological analysis of the spiral ligament of Slc4a10 knockout mice does not reveal overt fibrocyte loss as reported for Slc4a7 knockout mice. The ultrastructural analysis, however, shows mitochondrial alterations in fibrocytes of Slc4a10 knockout mice. Our data suggest that Slc4a10 and Slc4a7 are functionally related and essential for inner ear homeostasis.

Cochlear connexin 30 homomeric and heteromeric channels exhibit distinct assembly mechanisms.

Many of the mutations in GJB2 and GJB6, which encode connexins 26 and 30 (Cx26 and Cx30), impair the formation of membrane channels and cause autosomal syndromic and non-syndromic hearing loss. In cochlear non-sensory supporting cells, Cx26 and Cx30 form two types of homomeric and heteromeric gap junctions. The biogenesis processes of these channels occurring in situ remain largely unknown. Here we show that Cx30 homomeric and Cx26/Cx30 heteromeric gap junctions exhibit distinct assembly mechanisms in the cochlea. When expressed as homomeric channels, Cx30 preferentially interacts with ß-actin in the peripheral non-junctional membrane region, called perinexus, and strongly relies on the actin network for gap junction plaque assembly. In contrast, we found that Cx26/Cx30 heteromeric gap junction plaques are devoid of perinexus and associated actin network, and resist to actin-depolymerizating drug. This supports that Cx26/Cx30 oligomers could be directly delivered from the interior of the cell to the junctional plaque. Altogether, our data provide a novel insight in homomeric and heteromeric gap junction plaque assembly in the cochlea.

Site specific hypermethylation of CpGs in Connexin genes 30, 26 and 43 in different grades of glioma and attenuated levels of their mRNAs.

AIM: Gliomas, the intracranial tumours are considered the deadliest malignancies. The gap junctional Connexins (Cxs) that maintain cellular homeostasis perform a unique function in glial tumour suppression. However, the differential methylation patterns of Cxs were not revealed in glioma so far. The current study attempts to categorise promoter methylation of Cx30 and Cx26 and intron methylation of Cx43 in different grades of human glioma. MATERIALS AND METHODS: About 85 glioma patients with pathologically confirmed grades and 15 control brain tissues were recruited in the study. Bisulphite-PCR-Single Stranded Conformation analysis(SSCA), Bisulphite sequencing and MeDIP-qPCR were carried out to assess methylation status and Cx mRNA levels were also analysed to evaluate the effect of methylation. RESULTS: We found that promoter CpG islands(CpGs) reside in Sp1 and Ap2 sites of Cx30 and 26 were hypermethylated in high grades (HG) of glioma rather than low grades. The input % of both was significantly increased (p < 0.03) in progressive grades. Interestingly, Cx43 could exhibit a significant increase (p < 0.05) in input % only in grade IV. While, Cx30 and 26 mRNAs were downregulated according to their methylation status in progressive fashion with grades, Cx43 was downregulated irrespective of intron methylation. CONCLUSION: Thus, we suggest that the sites and extent of methylation of Cxs (30 and 26 but not in 43) are found to be altered. In different grades of glioma can provide better appreciation of the grade of the patient and might help in strategies based on epigenetic approaches.